This application is a U.S. national stage of International Application PCT/FI99/00898, filed Oct. 27, 1999 and published on May 18, 2000 in the English language.
This invention relates to the chemistry and applications of the metalloporphyrin. In particular it is related to novel photoluminescent metalloporphyrin compounds, their preparation and to the use of the said compounds as labelling reagents for biologically active molecules. This invention can be applied for example, in biomedical research and in-vitro diagnostics, in food industry, in chemical and pharmaceutical industry, in biotechnology as well as in environmental monitoring.
Many (bio)analytical systems utilise certain chemical compounds and species as sensitive and specific probes for the determination of specific analytes and parameters. In particular, photoluminescent dyes are frequently used for labelling of biomolecules in different analytical applications. Various photoluminescent dyes with different physical, chemical and spectral properties have been described, that suit a wide range of applications, such as staining of proteins, nucleic acids, cells, for use in microscopy, immunoassays and DNA hybridisation assays. The most common families of the dyes include fluoresceins, rhodamines, coumarines, cyanines and BODIPY dyes (=boron dipyrrylmethine dyes). Families of the dyes which enable high detection sensitivity include photoluminescent lanthanide chelates and phosphorescent metalloporphyrins. A large number of photoluminescent fluorescent dyes and their biomolecule conjugates, such as protein conjugates and nucleic acid conjugates, have been developed in recent years. Many photoluminescent dyes as well as the corresponding labelling reagents and biomolecule conjugates are available commercially (see for example R. P. Haugland. Handbook of fluorescent probes and research chemicals, 6th Edn., Molecular Probes Inc., 1996) and widely used for diagnostics and research purposes. Photoluminescent conjugates of biomolecules are usually prepared by contacting a native biomolecule or a derivative of the biomolecule with a reactive derivative of a photoluminescent dye, also called labelling reagent. Examples of reactive groups in common labelling reagents include reactive esters (for example succinimidyl ester, sulphosuccinimidyl esters), isothiocyanato, chlorosulphonato, dichlorotriazinyl and maleimidyl groups. They enable simple and selective labelling of biomolecules via certain functional groups, most commonly via primary amino, thiol or hydroxy groups. Labelling can be performed under mild conditions and without using additional reagents. The availability of such standard labelling reagents and the corresponding photoluminescent bioconjugates is a requirement for development of analytical and diagnostic methods and applications. In many cases it is desirable to covalently attach the dye molecule via a spacer arm (usually 2-20 atoms long), in order to minimise the photochemical and biochemical interactions between the label and the biomolecule.
For those applications where high detection sensitivity is required, such as immunoassays and nucleic acid hybridisation assays, probes with long Stokes"" shift and well resolvable spectral characteristics are needed. Significant improvement of sensitivity, especially when working with complex biological samples, can be achieved by using long decay time photoluminescent dyes in combination with time-resolved photoluminescent detection. The time-resolved detection concept allows significant reduction of background interference from scattering and intrinsic fluorescence and consequently provides high signal-to-noise ratio (E. Soini et al. U.S. Pat. No 4,374,120, 1983).
Long decay time photoluminescent lanthanide chelates, in particular the complexes of Eu, Th, Sm, Dy have been suggested for use in sensitive time-resolved fluoroimmunoassays (for example U.S. Pat. No. 4,565,790, U.S. Pat. No. 5,346,996, U.S. Pat. No. 5,571,897). They are presently used in a commercial time-resolved fluoroimmunoassay system DELFIA(copyright) (trademark of EGandG-Wallac, Turku, Finland). p-isothiocyanatobenzyl-diethylenetriamine-N1,N2,N3,N4-tetraacetic acid chelated with Eu3+ is now commercially available for labelling of biomolecules, such as proteins, antibodies and nucleic acids.
Fluorescent complexes of ruthenium(II), osmium (II), and some other metals were suggested recently for use as long decay time fluorescent labels, particularly for oxygen sensors and for sensitive fluorescence immunoassays (J. Lakowicz et al., U.S. Pat. No. 5,660,991). Succinimidyl derivatives of Ruthenium bis(2,2xe2x80x2-bipyridine) (2,2xe2x80x2bipyridine-4,4xe2x80x2-dicarboxylic acid) have been described as labelling reagents for electrochemiluminescence immunoassays (U.S. Pat. No. 5,310,687).
A number of photoluminescent porphyrin dyes have been suggested for labelling of biomolecules, particularly for immunoassays (Schmidt D. et al. U.S. Pat. No. 4,614,723; Hendrix J., U.S. Pat. No. 4,707,454; Hendrix J., U.S. Pat. No. 5,464,741; Savitskii A. P. et al., Doklady Akademii Nauk SSSR, 1987, vol. 293, p.744, Braman, J. WO 96/11937). These dyes exhibit intense absorption bands around 400 nm and moderate absorption bands between 500 and 600 nm. Free base porphyrins usually emit fluorescence in red range of visible spectrum, whereas their metal complexes, mainly platinum(II) and palladium(II) porphyrins, exhibit bright phosphorescence in the same wavelength range even at room temperature (D. Dolphin, The Porphyrins, 1978, New York, Academic Press, vol. 3). Such properties make the porphyrin dyes promising in fluorescent and phosphorescent probing of biomolecules and particularly in time-resolved phosphorescence bioaffinity assays. A number of porphyrins as well as certain related structures, mainly biogenic polycarboxy porphyrins and water-soluble derivatives of tetraphenylporphyrin, have been suggested for such applications.
Water-soluble coproporphyrin-I was suggested by Savitsky et al. (Doklady Akademii Nauk SSSR, 1987, vol. 293, p.744) as a label for fluoroimmunoassays. A palladium(II) complex of coproporphyrin-I has been used for phosphorescence labelling of antibodies (Doklady Akademii Nauk SSSR 1989, vol. 304, p. 1005). In similar way some other polycarboxy porphyrins, including coproporphyrin-III, zinc(II)-coproporphyrin-I, dimethoxydeuteroporphyrin IX, hematoporphyrin IX, platinum(II)-coproporphyrin I and III, have been used for labelling various proteins. With these dyes covalent binding to proteins was usually achieved by carbodiimide method with various modifications. In a two-step procedure the carboxy groups of the porphyrin were first activated by carbodiimide in aqueous solution or in organic media and then allowed to react with protein amino groups in slightly alkaline aqueous solution. A drawback of the methods where carbodiimides are used for activation of porphyrins having more than one carboxy group, is that a mixture of different products is formed as a result of the carbodiimide activation. The mixture comprises porphyrins having from 0 to n pieces of activated carboxy groups where n is the number of carboxy groups in the porphyrin before the activation. Multi-point activation of porphyrins often causes significant crosslinking and inactivation of biomolecules such as proteins when they react with such reagents.
An additional drawback of the carbodiimide chemistry is that it does not allow the active product to be isolated in pure form, but the product remains in form of undefined mixture. Furthermore, the active carbodiimide adduct is instable. It is sensitive to hydrolysis as well as to many other nucleophiles. Typically, the carbodiimide adducts react intramolecularly producing non-reactive N-acylurea. The chemical instability of the carbodiimide-activated porphyrin adducts makes their use difficult and unreliable. Also porphyrin-antibody conjugates prepared by carbodiimide mediated coupling has been found to exhibit poor storage stability (Martsev et al., J.Immunol. Methods, 1995, vol. 186, p.293). The use of tetrasuccinimidyl esters of coproporphyrins as labelling reagent did not provide significant improvement in performance or stability of the conjugates (Martsev et al., J.Immunol. Methods, 1995, vol. 186, p.293).
In U.S. Pat. No. 4,614,723 Schmidt et. al. introduced the use of porphyrins comprising several carboxy groups as label for immunoassays. The method of activation of porphyrins according to Schmidt""s patent follows same carbodiimide chemistry as described above. G. Sagner and R. Haas (EP 0811626 A1) describe a preparation method for labelling reagents from water-soluble platinum(II) porphyrins: platinum coproporphyrin-I (PtCP) and platinum tetra-(p-carboxyphenyl) porphine (PtTCPP). However, in the patent publication as well as in two other publication by R Haas (J. Histochem. Cytochem., vol. 45, p.1279 (1997), J. Histochem. Cytochem, vol. 47,183-196 (1999)) the detailed chemical structures and respective systematic names of the label remain ambiguous. For example, the systematic name of a compound which they trivially call as xe2x80x98platinum coproporphyrinxe2x80x99, has been written as xe2x80x98Pt-3,8,13,18-tetramethyl-21H,23H porphine-2,7,12,17-tetrapropionic acidxe2x80x99. It is evident to a skilled person in the art, that upon insertion of a metal cation to the porphyrin cavity, a substitution occurs where the two hydrogen atoms bound to the positions 21 and 23 in the free base porhyrins are replaced. Thus, in case the authors mean phosphorescent metallo porphyrin chelate, the systematic name for the product should be xe2x80x98Platinum (II) complex of 2,7,12,17-tetramethyl-21H,23H-porphine-3,8,13,18-tetrapropionic acidxe2x80x99. In EP 0811626A1, Sagner et. al., the labelling reagent was prepared by applying carbodiimide chemistry as applied in several other publications referred to above. The specific teaching of this Sagner""s patent publication is that the activation reaction of the hydrophilic carboxy porphyrin has been carried out in dry organic solvent where hydrolysis of the activated derivatives should not take place. Preparation of the active porphyrin derivative is carried out by treating carboxy group containing porphyrins with a mixture of carbodiimide, N-hydroxy benzothiazole (=HOBt) and N-hydroxy succinimide in organic solvent, usually dimethylformamide. The reactive product formed in this process was used as such, without any purification or isolation of the reactive species. Since in such an activation process a mixture of undefined and unstable products is formed, the isolation of a single reactive species in pure form is very difficult. An additional drawback of the method is that the multiple activated porphyrins formed in the process may cause crosslinking and subsequent inactivation of the bioactive molecules.
Overall, a general problem of the carbodiimide mediated conjugation, when applied to polycarboxy porphyrins, is the lack of selectivity (i.e. all carboxy groups are subject to activation). Another problem is the formation of a complex mixture of labile compounds at the very first chemical stage of the conjugation reaction. For such mixtures it is difficult to apply conventional separation methods, so as to isolate in a pure form individual monofunctional reactive derivative(s), which can be characterised and then used for labelling of biomolecules.
In U.S. Pat. No. 5464741, U.S. Pat. No. 4,707,454 and in EP 0071991 Hendrix describes the use of porphyrins as label. U.S. Pat. No. 5,464,741 deals with the use of palladium(II) octaethylporphine alpha-isothiocyanate as a phosphorescent label for immunoassays. This monofunctional reagent is highly hydrophobic and not water-soluble. Consequently, it has limited use in labelling of biomolecules. An additional drawback of the reagents described by Hendrix is that the reactive group is bound directly next to the porphyrin moiety without any spacer arm. As usual, the lack of spacer arm has a tendency to significantly reduce the performance of the label. Furthermore, the same confusion with structures and names as found in the patent of Sagner et al., is also present in Hendrix""s patent. The chemical structures of the compounds of issue remain ambiguous. Since the compound presented by Hendrix are octaalkyl porphyrins, which are not dealt in this invention, the Hendrix""s patent is out of the scope of this invention.
In U.S. Pat. No. 4,707,545 and EP 0071991 Hendrix describes the use of chlorophyll and porphyrin derivatives as labels for fluoroimmunoassay. The key point in these two patents is the utilisation of the characteristic long Stokes"" shift of chlorophyll probes for improving the detection sensitivity of the assay. The coupling of the chlorophyll derivatives to an active assay reagent is carried out by means of in situ activation. No chlorophyll labelling reagent has been isolated. Since these two patents deal only with fluorescent markers, they are out of the scope of present invention.
Shoichet et. al. (Siberian Chem. J. 4, 32, 1991) describes the use of palladium (II) complex of 5-isothiocyanotobenzamino octaethylporphine for labelling of oligonucleotides. This monofunctional labelling reagent is highly hydrophobic and not water-soluble, and consequently has limited use in labelling of biomolecules.
Braman et. al. (WO 96/11937) describes use of tetraphenylporphyrins for labelling of nucleosides, nucleotides, oligonucleotides and polynuleotides. According to Braman""s publication, the porphyrin moiety on the labelled compound is detected on the basis of a reaction product produced by a porphyrin catalysed oxidation reaction. Said oxidation reaction may result in the formation of light, i.e., a chemiluminescent reaction, or a coloured compound, i.e., a colorimetric reaction. Since the Braman""s publication deals with tetraphenylporphyrins which are not dealt in the present invention, and since detection in Braman""s publication is not based on phosphorescence as in the present invention but on colorimetry and chemiluminescent, is the Braman""s publication out of the scope of the present invention.
The characteristic features of this invention are disclosed in the claims. The present invention describes novel phosphorescent metalloporphyrin compounds, their preparation and the use of said compounds as labelling reagents for preparation of phosphorescent conjugates with biomolecules. The labelling reagents obtainable according to this invention are preferably water soluble monofunctional derivatives of Pt- and Pd-coproporphyrins. The term xe2x80x98monofunctionalxe2x80x99 indicates that one of the four originally equal carboxy containing side chains of coproporhyrin has been differentiated and can be selectively further reacted. This invention offers also a route for preparation of such labelling reagents as well as the key intermediate compounds in a pure form, in other words, in form of stable organic compounds having defined chemical structure and properties. The compounds can be used as labelling reagents for labelling of biomolecules like peptides, proteins, hormones, nucleic acids, for labelling of cells, tissue or other chemical species having at least one functional group suitable for labelling reaction. The labelling reaction takes place under mild and slightly alkaline condition and can be carried out by any skilled person in the art. This invention also describes preparation of various bioconjugates using said metalloporphyrins as labelling reagents and the properties and the use of the such bioconjugates. The invention also describes the use of such bioconjugates in high sensitivity bioaffinity assays based on time-resolved phosphorescence. The bioaffinity assay may be e.g. an immunoassay, nucleic acid hybridisation and it can be performed in solution or on a solid substrate and the sample can be analysed with a time-resolved fluorometer/phosphorimeter. The assay can also be microscopic investigation of an immunocytological or immunohistological specimen.
The chemistry in the field of the use of the labelling reagents according to this invention most often takes place in aqueous media. Consequently, an applicable label needs to be water soluble and preferably hydrophilic. However, the aromatic tetrapyrrole unit i.e. porphin found in all porphyrins is hydrophobic in nature and not water soluble. In order to make the porphin unit water soluble, it must be substituted with hydrophilic side groups. One of the most preferable groups for this purpose is the carboxy group. In this context the term xe2x80x98carboxy groupxe2x80x99 means the chemical group that equals with the chemical formula xe2x80x94COOH, i.e. the group that corresponds to carboxylic acids. On the other hand, the term xe2x80x98oxycarbonyl groupxe2x80x99 means the chemical group that equals with the chemical formula xe2x80x94COOxe2x80x94, i.e. the diyl component found in carboxy group as well as in alkoxycarbonyl groups. The carboxy group is anionic in nature and has a tendency to make otherwise hydrophobic species water soluble in neutral and alkaline pH. Therefore, the most promising porphyrin compounds for labelling of biomolecules are those having carboxy (or the corresponding carboxylate) functional groups as substituents on the porphin periphery. For example, coproporphyrins, which contains carboxy groups, are sufficiently water-soluble and can be used as a label in aqueous media. In context to this text the term coproporphyrin includes both free bases as well as metallocomplexes of coproporhyrin I and/or II.
A basic requirement for a commercially valuable labelling reagent is the monofunctionality with respect to the number of chemically reactive groups in the labelling reagent. This feature enables conjugation with biomolecules even in high target molecule concentration and high reagent concentration without causing deleterious cross-linking. The use of high concentrations makes labelling process more reliable and produces a product with higher degree of label substitution.
A further requirement for a commercially valuable labelling reagent is that the method used for the preparation of said reagent must enable reasonably high synthetic yield. An additional requirement is that the method of preparation enables isolation of the labelling reagent in pure form, in other words, in the form of a stable organic compound having a defined chemical structure and defined properties.
To improve the label performance, it is desirable to have an additional spacer arm (usually 2 to 20 atoms long) between the reactive site and the chromophoric moiety of the reagent. The spacer arm can serve for example to reduce steric hindrance around the reactive site, to make the reactive site more mobile, and to reduce the physical interaction between the label and the biomolecule.
It can be seen that the phosphorescent porhyrin-based labelling reagents according to prior art, usually lack one or more of the preferred properties.
In present invention we describe novel phosphorescent Pd- and Pt-coproporphyrins which fulfil the preferred properties as discussed above and which can be used for facile labelling of biologically active molecules having one or more functional groups suitable for the conjugation reaction. General structures that are characteristic for the labelling reagents which essentially fulfil the above mentioned requirements, are shown in Scheme I (Structure I and Structure II). In these compounds M is palladium (II) or platinum (II), R1 is xe2x80x94OH, or xe2x80x94Oxe2x88x92X+ group where X is a cation, most usually alkali metal cations or ammonium cations and R2 is a chemical moiety bearing a linker unit xe2x80x94Yxe2x80x94 and a functional group xe2x80x94Z where said functional xe2x80x94Z group can be used for selective covalent linkage to other molecules, such as biomolecules, and/or for further chemical modifications. The linker unit xe2x80x94Yxe2x80x94 is either a covalent bond or a C1-C20 straight or branched alkylene, arylene, alkarylene or aralkylene group, which also may contain heteroatoms or heteroatoms containing side chains, or cyclic residues. The linker unit may also comprise or be composed of residues of polymers, preferably residues of hydrophilic polymers such as residues of polypeptides, polysaccarides, polynucleotides, polyethers or other. Among the most preferable chemical moieties xe2x80x94YZ are, according to this invention, those having a general formula xe2x80x94(CH2)nxe2x80x94Phxe2x80x94Z, where xe2x80x94Z is either amino (xe2x80x94NH2) or isothiocyanato (xe2x80x94NCS) group, xe2x80x94Phxe2x80x94 is a phenylene group, and n is an integer from 1 to 10.
From the Structure I and Structure II (Scheme I) one can see that the main object of this invention is the differentiation and subsequent functionalisation of one single oxycarbonyl group in coproporphyrins used as starting material. Such differentiation is realised via selective hydrolysis of the appropriate tetraalkyl esters of corresponding coproporphyrins, by using simple chemical procedure and technique.
Although it is clear that simple hydrolysis of tetraesters of coproporphyrins (as well as other polyesters of porphyrins) usually results in number of products, the present invention demonstrates that, if the hydrolysis reaction is carried out under the particular conditions described in this invention, surprisingly high amount of the coproporphyrin monoacid can be produced. After a simple chromatographic separation, a pure monocarboxy product can be isolated, whereas all the other by-products can be pooled and then transformed back into the form of tetraester by simple reesterification. Such reesterification reaction usually proceeds smoothly and almost quantitatively, thus saving most of the raw material which then can be recycled. Considering easy and simple recycling, the net yield of target monoacid of coproporphyrin may approach its theoretical limit.
The monoacid of coproporphyrin is the key compound for further monofunctionalisation and preparation of labelling reagents and bioconjugates according to this invention. This compound obtained in a pure form can easily be modified into the form of appropriate monofunctionalised derivative(s), labelling reagent(s) or a precursor of a labelling reagent. The modification of the carboxylic acid residue in coproporphyrin monoacid can be performed easily by applying the conventional chemistry related to modification of carboxy groups. The carboxy group of coproporphyrin monoacid can be modified, so as to introduce various functional groups via this site using a variety of available chemical methods.
According to this invention, the preferred methods of monofunctionalisation of the monoacid of coproporphyrins include, for example, activation of the carboxy group for subsequent coupling reactions using methods that involve succinimide esters, sulfosuccinimide esters, pentafluorophenyl esters, 4-nitrophenol esters, N-hydroxy benzotriazole esters, carbodiimide adducts and others.
If further lengthening is desirable, an additional spacer may be attached to the differentiated side chain. This can be carried out by using appropriate compounds such as heterobifunctional linkers. The preferable heterobifunctional compounds according to this invention include, for example, those in which the two functional groups are an aliphatic primary amino group and an aromatic primary amino group. This class of linker compounds is particularly preferred in the scope of this invention because of following reasons. First, due to the remarkable difference in reactivities between the two amino groups, the coupling with an active ester proceeds without a risk of deleterious crosslinking. Second, said class of linkers compounds comprise an inherent precursor for the reactive isothiocyanato group. However, the scope of this invention is not limited to the use of said class of linker compounds, but any of linker compounds, such as homobifunctional linker compounds and polyfunctional linker compounds can also be applied.
The next step in the synthetic scheme of a coproporphyrin labelling reagent is the transformation of relatively hydrophobic triester of the monofunctionalised coproporphyrin into a water-soluble form by alkaline hydrolysis of the remaining alkoxycarbonyl groups. As described above, the three carboxy substituents provide a structure with solubility in neutral and alkaline aqueous solutions. This stage is expected to preserve the introduced functional group, the spacer and the main structure of the coproporphyrin derivative from undesirable transformations. At this stage a water-soluble derivative of coproporphyrin is obtained which bears three carboxy groups and one differentiated side chain. The differentiated side chain that comprises a spacer unit and a reactive chemical group, such as amino or hydroxy, can be used for covalent coupling as such or after further chemical modification. For example, a coproporphyrin having an aromatic primary amino group, can be used for covalent coupling as such, or in the form of its conversion product isothiocyanate, which is obtained from said aromatic primary amino group by treatment with thiophosgene. Corresponding phenyl-isothiocyanato derivatives of phosphorescent Pt- and Pd-coproporphyrins are preferred target compounds and labelling reagents according to this invention. However, the scope of this invention is not limited to the use of isothiocyanato group as final reactive group, but other chemical groups can also be applied.
In certain cases, where covalent labelling of a target molecule in organic solvent is preferred, the non-water soluble form of a differentiated coproporhyrin, i.e. coproporphyrin comprising up to three alkoxycarbonyl groups, may also be applied as a labelling reagent. Scheme I (Structure I and Structure II) can also be used to illustrate the general structures of such compounds. In this case M is palladium (II) or platinum (II), R1 is xe2x80x94OR group where R is C1-C20 straight or branched alkyl, aryl, alkaryl or aralkyl group, R2 is a chemical moiety bearing a linker unit xe2x80x94Yxe2x80x94 and a functional group xe2x80x94Z where said functional xe2x80x94Z group can be used for selective covalent linkage to other molecules, such as biomolecules, and/or for further chemical modifications. The linker unit xe2x80x94Yxe2x80x94 is either a covalent bond or a C1-C20 straight or branched alkylene, arylene, alkarylene or aralkylene group, which also, may contain heteroatoms or heteroatoms containing sidechains or cyclic residues. The linker unit may also comprise or be composed of residues of polymers, preferably residues of hydrophilic polymers such as residues of polypeptides, polysaccarides, polynucleotides, polyethers or other. Among the most preferable chemical moieties xe2x80x94YZ are, according to this invention, those having a general formula xe2x80x94(CH2)nxe2x80x94Phxe2x80x94Z, where xe2x80x94Z is either amino (xe2x80x94NH2) or isothiocyanato (xe2x80x94NCS) group, xe2x80x94Phxe2x80x94 is a phenylene group, and n is an integer from 1 to 10.
The phosphorescent labelling reagents described above such as water-soluble phenyl-isothiocyanate derivatives of Pt- and Pd-coproporphyrins, can be used for facile labelling of various biomolecules such as peptides, proteins, haptens, nucleic acids; labelling of cells, tissue or other chemical species. As products of such labelling reactions the phosphorescent conjugates can be obtained, which are also the object of this invention. Said conjugates can be used as probes in sensitive bioaffinity assays, which are based on bioaffinity reactions and time-resolved phosphorescence detection.